COPPER-DRIVEN REDOX MODULATION AMPLIFIES THYMOQUINONE- MEDIATED DNA DAMAGE, APOPTOSIS SIGNALING, AND TUMOR SUPPRESSION IN COLORECTALCANCER

Copper (Cu) dysregulation has emerged as a hallmark of colorectal cancer (CRC), yet its therapeutic exploitation remains underexplored. In this project, we investigated whether Cu availability enhances the anticancer efficacy of phytochemical thymoquinone (TQ) through oxidative stress-mediated mechanisms. Our results show that Cu supplementation sensitized resistant HT-29 CRC cells to TQ, potentiating intracellular ROS generation, enhanced apoptosis, and DNA damage, as evidenced byelevatedγH2A levels and downregulation of the anti-apoptotic protein XIAP. In contrast, Cu supplementation did not further enhance TQ cytotoxicity in HCT-116 CRC cells, consistent with their elevated basal Cu levels, highlighting tumor-specific Cu dependence. Importantly, Cudid not enhance the cytotoxicity of TQ on non-tumorigenic intestinalFHs74Int cells, indicating a cancer-selective activity. Mechanistically, we showed that TQ can redox-cycle Cu(II) to Cu(I) in a dose-dependent manner, identifying a Cu-dependent oxidative stress response.HT-29 xenografts revealed that Cu supplementation significantly potentiated TQ-mediated tumor suppression, promoting a 35%further reduction in tumor volume compared to TQ alone. Histological evaluation using H & E staining showed a marked decrease in malignant nuclear staining in tumors treated with Cu and TQ, consistent with the observed tumor growth inhibition. Collectively, these findings highlight that Cu availability amplifies TQ-induced oxidative stress mechanisms, providing a mechanistic basis for enhanced tumor suppression. Targeting Cu metabolism alongside natural phytochemicals such as TQ holds a promising strategy for enhancing therapeutic response and inhibiting CRC progression.